Control of plant virus diseases took a major step forward in the last decade when it was shown in 1986 that the tobacco mosaic virus (“TMV”) coat protein gene that was expressed in transgenic tobacco conferred resistance to TMV (Powell-Abel, P., et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science, 232:738-43 (1986)). The concept of pathogen-derived resistance (“PDR”), which states that pathogen genes that are expressed in transgenic plants will confer resistance to infection by the homologous or related pathogens (Sanford, J. C., et al. “The Concept of Parasite-Derived Resistance—Deriving Resistance Genes from the Parasite's Own Genome,” J. Theor. Biol., 113:395-405 (1985)) was introduced at about the same time. Since then, numerous reports have confirmed that PDR is a useful strategy for developing transgenic plants that are resistant to many different viruses (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Photopathol., 33:323-43 (1995)).
Only eight years after the report by Beachy and colleagues (Powell-Abel, P., et al., “Delay of Disease Development in Transgenic Plants that Express the Tobacco Mosaic Virus Coat Protein Gene,” Science, 232:738-43 (1986)), Grumet, R., “Development of Virus Resistant Plants via Genetic Engineering,” Plant Breeding Reviews, 12:47-49 (1994) reviewed the PDR literature and listed the successful development of virus resistant transgenic plants to at least 11 different groups of plant viruses. The vast majority of reports have utilized the coat protein genes of the viruses that are targeted for control. Although the testing of transgenic plants have been largely confined to laboratory and greenhouse experiments, a growing number of reports showed that resistance is effective under field conditions (e.g., Grumet, R., “Development of Virus Resistant Plants via Genetic Engineering,” Plant Breeding Reviews, 12:47-49 (1994)). Two virus resistant crops have been deregulated by APHIS/USDA and thus are approved for unrestricted release into the environment in the U.S.A. Squash that are resistant to watermelon mosaic virus 2 and zucchini yellow mosaic potyviruses have been commercialized (Fuchs, M., et al., “Resistance of Transgenic Hybrid Squash ZW-20 Expressing the Coat Protein Genes of Zucchini Yellow Mosaic Virus and Watermelon Mosaic Virus 2 to Mixed Infections by Both Potyviruses,” Bio/Technology, 13:1466-73 (1995); Tricoli, D. M., et al., “Field Evaluation of Transgenic Squash Containing Single or Multiple Virus Coat Protein Gene Constructs for Resistance to Cucumber Mosaic Virus, Watermelon Mosaic Virus 2, and Zucchini Yellow Mosaic Virus,” Bio/Technology, 13:1458-65 (1995)). Also, a transgenic papaya that is resistant to papaya ringspot virus has been developed (Fitch, M. M. M., et al., “Virus Resistant Papaya Derived from Tissues Bombarded with the Coat Protein Gene of Papaya Ringspot Virus,” Bio/Technology, 10:1466-72 (1992); Tennant, P. F., et al., “Differential Protection Against Papaya Ringspot Virus Isolates in Coat Protein Gene Transgenic Papaya and Classically Cross-Protected Papaya,” Phytopathology, 84:1359-66 (1994)). This resistant transgenic papaya was recently deregulated by USDA/APHIS. Deregulation of the transgenic papaya is timely, because Hawaii's papaya industry is being devastated by papaya ringspot virus. Undoubtedly, more crops will be deregulated and commercialized in the near future.
Interestingly, remarkable progress has been made in developing virus resistant transgenic plants despite a poor understanding of the mechanisms involved in the various forms of pathogen-derived resistance (Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Photopathol., 33:323-43 (1995)). Although most reports deal with the use of coat protein genes to confer resistance, a growing number of reports have shown that viral replicase (Golemboski, D. B., et al., “Plants Transformed with a Tobacco Mosaic Virus Nonstructural Gene Sequence are Resistant to the Virus,” Proc. Natl. Acad. Sci. USA, 87:6311-15 (1990)), movement protein (e.g., Beck, D. L., et al., “Disruption of Virus Movement Confers Broad-Spectrum Resistance Against Systemic Infection by Plant Viruses with a Triple Gene Block,” Proc. Natl. Acad. Sci. USA, 91:10310-14 (1994)), NIa proteases of potyviruses (e.g., Maiti, I. B., et al., “Plants that Express a Potyvirus Proteinase Gene are Resistant to Virus Infection,” Proc. Natl. Acad. Sci. USA, 90:6110-14 (1993)), and other viral genes are effective. This led to the conclusion that any part of a plant viral genome gives rise to PDR. Furthermore, the viral genes can be effective in the translatable and nontranslatable sense forms, and less frequently antisense forms (e.g., Baulcombe, D. C., “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell, 8:1833-44 (1996); Dougherty, W. G., et al., “Transgenes and Gene Suppression: Telling us Something New?,” Current Opinion in Cell Biology, 7:399-05 (1995); Lomonossoff, G. P., “Pathogen-Derived Resistance to Plant Viruses,” Ann. Rev. Photopathol., 33:323-43 (1995)).
RNA-mediated resistance is the form of PDR where there is clear evidence that viral proteins do not play a role in conferring resistance to the transgenic plant. The first clear cases for RNA-mediated resistance were reported in 1992 for tobacco etch (“TEV”) potyvirus (Lindbo, et al., “Pathogen-Derived Resistance to a Potyvirus Immune and Resistance Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,” Mol. Plant Microbe Interact., 5:144-53 (1992)), for potato virus Y (“PVY”) potyvirus by Van Der Vlugt, R. A. A., et al., “Evidence for Sense RNA-Mediated Protection to PVY in Tobacco Plants Transformed with the Viral Oat Protein Cistron,” Plant Mol. Biol., 20:631-39 (1992), and for tomato spotted wilt (“TSWV”) tospovirus by de Haan, P., et al., “Characterization of RNA-Mediated Resistance to Tomato Spotted Wilt Virus in Transgenic Tobacco Plants,” Bio/Technology, 10:1133-37 (1992). Others confirmed the occurrence of RNA-mediated resistance with potyviruses (Smith, H. A., et al., “Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,” Plant Cell, 6:1441-53 (1994)), potexviruses (Mueller, E., et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal, 7:1001-13 (1995)), and TSWV and other topsoviruses (Pang, S. Z., et al., “Resistance of Transgenic Nicotiana Benthamiana Plants to Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses: Evidence of Involvement of the N Protein and N Gene RNA in Resistance,” Phytopathology, 84:243-49 (1994); Pang, S.-Z., et al., “Different Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt Virus and Impatiens Necrotic Spot Tospoviruses,” Bio/Technology 11:819-24 (1993)). More recent work has shown that RNA-mediated resistance also occurs with the comovirus cowpea mosaic virus (Sijen, T., et al., “RNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,” Plant Cell, 8:2227-94 (1996)) and squash mosaic virus (Jan, F.-J., et al., “Genetic and Molecular Analysis of Squash Plants Transformed with Coat Protein Genes of Squash Mosaic Virus,” Phytopathology, 86:S16-17 (1996)).
Major advances towards understanding the mechanism(s) of RNA-mediated resistance were made by Dougherty and colleagues in a series of experiments with TEV and PVY. Using TEV, Lindbo, J. A., “Pathogen-Derived Resistance to a Potyvirus Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,” Mol. Plant Microbe Interact., 5:144-53 (1992) and Lindbo, J. A., et al., “Untranslatable Transcripts of the Tobacco Etch Virus Coat Protein Gene Sequence can Interfere with Tobacco Etch Virus Replication in Transgenic Plants and Protoplasts,” Virology, 189:725-33 (1992) showed that transgenic plants expressing translatable full length coat protein, truncated translatable coat protein, antisense coat protein genes, and nontranslatable coat protein gene had various phenotypic reactions after inoculation with TEV. Transgenic plants displayed resistance, recovery (inoculated plants initially show systemic infection but younger leaves that develop later are symptomless and resistant to the virus), or susceptible phenotypes. Furthermore, they showed that leaves of resistant plants and asymptomatic leaves of recovered plants had relatively low levels of steady state RNA when compared to those in leaves of susceptible plants (Lindbo, J. A., et al., “Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,” Plant Cell, 5:1749-59 (1993)). However, nuclear run off experiments showed that those plants with low levels of steady state RNA had higher transcription rates of the viral transgene than those plants that were susceptible (and had high steady state RNA levels). To account for these observations, it was proposed “that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation” (Lindbo, J. A., et al., “Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,” Plant Cell, 5:1749-59 (1993)). It was also suggested that the low steady state RNA levels may be due to post-transcriptional gene silencing, a phenomenon that was first proposed by de Carvalho, F., et al., “Suppression of beta-1,3-glucanase Transgene Expression in Homozygous Plants,” EMBO J., 11:2595-602 (1992) for the suppression of β-1,3-glucanase transgene in homozygous transgenic plants.
An RNA threshold model was proposed to account for the observations (Lindbo, J. A., et al., “Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance,” Plant Cell, 5:1749-59 (1993)). Basically, the model states that there is a cytoplasmic cellular degradation mechanism that acts to limit the RNA levels in plant cells, and that this mechanism is activated when the transgenic RNA transcript goes above a threshold level. The degradation mechanism is specific for the transcript that goes above the threshold level; and if the transcripts that go above a certain threshold is a viral transgene, the virus resistance state is observed in the plant, because the degradation mechanism also targets, for inactivation, the specific sequences of the incoming virus. The model also accounts for the ‘recovery’ of transgenic plants by suggesting that viral RNA from the systemically invading virus triggers the phenomenon in some transgenic plants that have two copies of the transgenes. Plants that had more than three copies of the transgenes caused the threshold level to be surpassed without the invasion of virus (Goodwin, J., et al., “Genetic and Biochemical Dissection of Transgenic RNA-Mediated Virus Resistance,” Plant Cell, 8:95-105 (1996); Smith, H. A., et al., “Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs: Expression, Regulation, and Fate of Nonessential RNAs,” Plant Cell, 6:1441-53 (1994)). Although the degradation mechanism is not clear, it is proposed that a cellular RNA dependent RNA polymerase (“RdRp”) binds to the transcript and produces small fragments of antisense RNA which then bind to other transcripts to form duplexes which are then degraded by nucleases that specifically recognize RNA-RNA duplexes. This degradation mechanism is sequence specific, which accounts for the specificity of RNA-mediated resistance.
Work on PVX by Baulcombe and colleagues (English, J. J., et al., “Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes,” Plant Cell, 8: 179-88 (1996); Mueller, E., et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal, 7:1001-13 (1995)) confirmed and extended the results by Dougherty and colleagues. An aberrant RNA model which is a modification of the RNA threshold model proposed by Dougherty and colleagues was proposed. The features of the model are similar to Dougherty's except that it states that the RNA level is not the sole trigger to activate the cellular degradation mechanism, but instead aberrant RNA that are produced during the transcription of the transgene plays an important part in activating the cytoplasmic cellular mechanism that degrades specific RNA. The production of aberrant RNA may be enhanced by positional affects of the transgene on the chromosome and by methylation of the transgene DNA. The precise nature of the aberrant RNA is not defined, but it may contain a characteristic that makes it a preferred template for the production of antisense RNA by the host encoded RdRp (Baulcombe, D. C., “Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants,” Plant Cell, 8:1833-44 (1996); English, J. J., et al., “Suppression of Virus Accumulation in Transgenic Plants Exhibiting Silencing of Nuclear Genes,” Plant Cell, 8: 179-88 (1996)). Thus, the model also proposes that RdRp and antisense molecules are involved in the degradation mechanism. Baulcombe and colleagues confirmed that plants which show low steady state transgene levels have multiple copies of transgenes and that the low steady state RNA and the accompanying resistant state is due to post-transcriptional gene silencing. The term homology-dependent resistance was proposed to describe the resistance in plants that show homology-dependent gene silencing (Mueller, E., et al., “Homology-Dependent Resistance: Transgenic Virus Resistance in Plants Related to Homology-Dependent Gene Silencing,” Plant Journal, 7:1001-13 (1995)).
Experiments with TSWV tospovirus (Pang, S. Z., et al., “Post-Transcriptional Transgene Silencing and Consequent Tospovirus Resistance in Transgenic Lettuce are Affected by Transgene Dosage and Plant Development,” Plant Journal, 9:899-09 (1996); Prins, M., et al., “Engineered RNA Mediated Resistance to Tomato Spotted Wilt Virus is Sequence Specific,” Mol. Plant Microbe Interact., 9:416-18 (1996)) and cowpea mosaic comovirus (Sijen, T., et al., “RNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,” Plant Cell, 8:2227-94 (1996)) also showed that resistance in transgenic plants is a consequence of post-transcriptional gene silencing. Pang, S. Z., et al., “Post-Transcriptional Transgene Silencing and Consequent Tospovirus Resistance in Transgenic Lettuce are Affected by Transgene Dosage and Plant Development,” Plant Journal, 9:899-09 (1996) showed that post-transcriptional gene silencing in transgenic lettuce expressing the N gene of TSWV was influenced by gene dosage and by the developmental stage of the plant. The effect of developmental stage on post-transcriptional gene silencing of transgenes and their effect on resistance had not been previously shown for transgenic plants expressing viral genes, but had been shown to occur in plants expressing other transgenes (de Carvalho, F., et al., “Suppression of beta-1,3-glucanase Transgene Expression in Homozygous Plants,” EMBO J., 11:2595-02 (1992)). Post-transcriptional gene silencing could also account for the correlation of low steady state level of N gene RNA in transgenic tobacco showing very high but specific resistance (Pang, S. Z., et al., “Different Mechanisms Protect Transgenic Tobacco Against Tomato Spotted Wilt and Impatiens Necrotic Spot Tospoviruses,” Bio/Technology, 11:819-24 (1993)). Prins, M., et al., “Engineered RNA-Mediated Resistance to Tomato Spotted Wilt Virus is Sequence Specific,” Molecular Plant Microbe Interactions, 9:416-18 (1996) also reported that post-transcriptional gene silencing occurred with transgenic tobacco expressing the N gene and nonstructural gene of the mRNA. Interestingly, it was found that tobacco with other parts of the TSWV genome were not resistant. They suggested, as one explanation, that those gene fragments which did not confer resistance may not fit the criteria for inducing post-transcriptional gene silencing. Sijen, T., et al., “RNA-Mediated Virus Resistance: Role of Repeated Transgene and Delineation of Targeted Regions,” Plant Cell, 8:2227-94 (1996) showed that resistance of transgenic plants expressing the movement protein, replicase, or coat protein were due to post-transcriptional gene silencing. This data also suggested that the 3′ region of the movement protein transgene mRNA is the initial target of the silencing mechanism.
The present invention is directed to producing improved disease resistant plants.